DDB Pull Down
Thaw ~2ml frozen brain lysate, rapid thaw at 37 until just small amount of frozen ice left. Gently turn the tube several times to mix during thawing, avoid bubbles. # add 2mM DTT (or TCEP) # add 0.1% NP40 from 10% stock # add 1mM PMSF # centrifuge x80 kg for 10 min, 4deg C, TLA 100.4 rotor (properly greased o-rings), be sure to lock rotor by pressing down on lid button. # add 60-80ul BicD2 (usually around 300nM final conc.) and 2uM SNAP-TMR (or other color) dye. Incubate on ice for 30-60 min to allow DDB complex formation and TMR labeling of BicD2. # Incubate 1hr with 400ul 50% sepharose strep resin (e.g. 200ul beads, 200ul wash buffer). NOTE: Use GE streptactin sepharose beads NOT the Qiagen fast flow beads we use in the large columns for protein preps. #* rotate 4 deg C 1hr. #* wash 5-7 times, 750ul wash buffer. Wash buffer: SRP, 1mM DTT, 0.1% NP-40, 1mM PMSF #* Wash by gentle resuspension of beads with buffer, I usually use pipette or turn tube over several times. After first spin, resuspend the beads and transfer to a 1.6mL tube. The conical bottom makes it easier to remove the supernatant. NO BUBBLES! Spin in small fuge for 10 sec. to pellet beads and aspirate buffer from top. #* Elute by incubation with Wash buffer including 3mM desthiobiotin (~200ul total elution, try to match the bead volume 1:1). Incubate with elution buffer on ice for at least 10-15 min. Gentle pipetting of beads a few times when they settle to bottom of tube. NOTE: You MUST be sure your elution buffer pH is above 7.0! Protein won’t elute below 7.0 and desthiobiotin can lower pH. Double check pH of elution buffer after dissolving desthiobiotin and adjust to 7.0 or a little higher! * Recover supernatant via spin filter with a milipore pvdf membrane 0.22uM filter size microcentrifuge tube. ** Immediately re suspend the dried beads in buffer. Add beads to the "dirty" beads tube in the fridge for future regeneration. ** Spin 30 sec. in room temp fuge at full speed (15K rpm). ** Quickly place eluted complex on ice. ** Take top of filter with dried strep beads and resuspend in 300uL elution buffer. Put in “dirty/used beads” tube for later regeneration. 8. Take eluted DDB complex and add sucrose to 10% final. Use 50% sucrose solution in 50mL falcon tube on bench. 50% sucrose is very viscous! Pipette carefully and it will sink to bottom of eluted DDB. Be sure to mix gently by pipetting until you no longer see sucrose swirling in the DDB buffer (hold to light while mixing). 9. Immediately freeze DDB complex in 5uL aliquots using liquid nitrogen. Use small colored pcr tubes, left side on the tube shelf near hood. Store at -80 in bag clearly labeled. Polymerize tubulin, separate poly from non-poly tubulin: # Make biotin/fluorescent/unlabeled tubulin mixture, can play with ratios. About one in every 10 tubulin will be labeled with either biotin or dye below. ## 5ul unlabeled tubulin (~12.5mg/ml, 100kDa/dimer) ## 2ul 1:10 diluted fluorescent tub ## 2ul 1:10 biotinylated tub (I usually use 4uL if I want the MTs stuck down well). ## 1ul GTP (100mM stock) -------------------------------------------------- 10ul total → incubate in 37 deg C water bath for 10-15 min CRITICAL!: MIX THE TUBULIN WELL. USE A SMALLER VOLUME AND PIPETTE UP AND DOWN SEVERAL TIMES TO BE SURE THE TUBULIN WILL POLYMERIZE HOMOGENOUSLY BEFORE YOU STABILIZE IT WITH TAXOL! # Make 40uM stock taxol diluted in BRB80 and warm in 37 water bath # Add 10ul 40uM taxol to Mt mix and incubate at 37 for at least 15min # Spin down on sucrose cushion: ## 150ul 25% sucrose, 5uM taxol in fresh tube ## Layer the whole MT mix on top very gently ## spin max speed (15K rpm) in tabletop for 10min ## aspirate supernatant with vacuum tip CAREFULLY! Can suck the MT pellet right out of tube if not careful. ## resuspend pellet (poly Mts) in BRB80 buffer, 5uM taxol. Use 50uL buffer. Pipette up and down using narrow tip until you no longer see MT chunks/flakes in buffer (hold up to light while pipetting). Preparing samples on coverslips: # Flow 20ul BSA-biotin through flow cell, wait 2-3 min (longer if you want really good sticking). ## Alternative: Use 20 ul of 0.5mg/mL PPL-PEG-Biotin instead of BSA-Biotin # Washing buffer “BC” is BRB80, 1mg/mL BSA, 1mg/mL Casein, 0.5% Pluronic F-168 # Be sure BC buffer aliquot has 5uM taxol in it. # Wash with 20ul BC buffer # Flow 20ul of streptavadin ## *good step to pause and put some chambers in # Wash 20uL BC buffer # Dilute 2ul Mts with 20ul of BC buffer, can change ratio accordingly ## wait ~10 min 8. Wash out unbound MTs with 20uL BC buffer. Check chamber on scope to see if density of MTs is good. If not, add more MTs and wait longer. If looks good, wash again with ASS buffer which will quench all the rest of the available streptavidin. * ASS Buffer: SRP buffer with: 5uM taxol, 0.5% Pluronic, 0.2mg/mL Kappa-casein, 0.1mg/mL biotin-BSA 9. Once you are satisfied with MT chamber, thaw a DDB aliquot from the -80. Quick thaw at 37 is good, will only take 5 sec. 10. Determine what dilution to use motor at. I usually try 1:50 to start, depends on prep: ## x ul DDB pull down (usually 1-5ul) ## 1ul 100mM ATP (final 1mM) ## 1ul PCA ## 0.5ul PCD ## 1ul Trollox ## to 50ul, ASS buffer * Flow in 2x20uL final motor mixture and go image immediately. Motors usually too dense at start of imaging, but start to thin out overtime as they accumulate at MT ends. Streptavidin-free protocol (Methyl cellulose) # Block chamber for 10 min with highly concentrated BSA. May be better to use BC buffer with BSA, casein and Pluronic. # Dilute MTs into BC buffer with 0.2% MeCell. Pipette to make sure MTs are evenly dispersed or they may bundle! Flow in your Mt dilution and allow MTs to diffuse to coverslip. Check by TIRF. # Wash with BC + 0.2% MeCell # Check Mts on scope to make sure they weren’t washed away # Flow in motor dilution with 0.2% MeCell ## Other components same as always Scope: “Don’t fuck up the objective” - Rick 2016 * Make sure scope is on and working before thawing any critical components like the DDB complex. Best way is to look at MTs first before doing anything.